The purposes of the experiments conducted within this paper are: 1) to successfully synthesize a compound that is inherent to over fifty percent of a library obtained from Find-A-Drug, 2) to attempt to make a Ugi product, 3) to monitor imine reversal and the technique to prevent reversal, and 4) to conduct research in an Open Source manner via Wikis and Blogs. The successful synthesis of the particular compound DOPAL was required because it is not readily commercially available. Compounds thought to inhibit enoyl reductase, an enzyme utilized by malarial parasites, were obtained from Find-A-Drug in a library of 250 possible diketopiperazines. This research is unbarred from normal constraints of laboratory research in order to promote public discussion of experiments, results, theories and hypothesis so ideas stay fresh. To do this, experimental results have been published on this wiki for free viewing. NMR spectra were saved in JCAMP format and, utilizing a program developed by Robert Lancashire, uploaded onto a Drexel server and linked to on the main experiment wiki page.
Introduction
This research it focused on developing an anti-malarial compound which was based on a library of similar probable compounds that were discovered by Find-A-Drug 1. While there are other anti-malarial drugs on the market, it was believed that a better anti-malarial could be made through new pathways. The reason for this is due to the powerful resistivity of the protozoan parasites that are classified into four species which cause malaria. The genus that all of the parasites have in common is called plasmodium and the four species are falciparum, vivax, ovale, and malaria 2. The compounds of this library are 1,4-diketopiperazines 3. Using software which mimics a bonding capabilities of compounds that are particularly complex including protein structures, these compounds were found to be good inhibitors to a particular enzyme found in malaria, enoyl reductase. The enoyl-acyl carrier protein reductase (ENR) is involved in bacterial fatty acid biosynthesis 4. It is believed that the inhibition of this protein would terminate the parasite’s ability to survive in the human blood stream. While it not a vaccine the compound may be a better substitute to some of the other anti-malarial drugs on the market.
Through a literature search, a one pot multi-component reaction was found to synthesize a starting compound 3. This reaction is the Ugi reaction which combines a protected amino acid, an aldehyde, an amine, and an isocyanide. These compounds were mixed in methanol and allowed to react for 24 hours. After this, the methanol would be evaporated and then the Ugi product would be treated with 10% TFA in 1,2 dichloroethane. The result according to Hulme et al 5 is a cyclization of the Ugi product into a 1,4-diketopiperazine. The particular groups attached to the piperazine ring could be varied by particular acids, amines and aldehydes but there was one particular substituent that was in over 50% of the library - a 4-ethyl-1,2-dihydroxyphenol. In accordance with the Ugi reaction this substituent starts out as an aldehyde this aldehyde was DOPAL. Unfortunately, DOPAL was not commercially available. Thus the work in this paper focuses on the making of DOPAL, the Ugi reaction, and trying to halt the reversal of imine formation of the aldehyde and amine after addition of the acid and isocyanide.
Another major facet of this research revolves around it being an Open Source research project. This open source project is generally defined as a free reference to all of our experiments, success or failure, that anyone, scientist or not, can comment on. Thus public discussion of experiments, results, theories, and discussions are encouraged and all data is available and up-to-date. To do this, experimental results have been published on a wiki including NMR spectra which were saved in JCAMP format and, utilizing a program developed by Robert Lancashire, uploaded onto a Drexel server and linked to on the main experiment wiki page. This way an individual has the ability to expand an area at will and does not have to rely on a scanner's resolution.
Experimental
DOPAL Procedure
The Compound DOPAL was synthesized from the reaction done by Robins et al 6. A solution of adrenaline in 85% phosphoric acid was heated in via a Glycerol bath. The vial was placed into the bath at 100C and then heated to 120C for 15 min then removed from heat and allowed to cool for 30 seconds. The solution was added to distilled water and allowed to sit for 1.5 hours. It was then saturated with NaCl. The solution was decanted off of the excess sodium chloride into a separatory funnel. The aldehyde was then extracted with ethyl ether, which were combined and dried over MgSO4. After filtration the solution was evaporated to yield crude DOPAL. See Exp 016 for exact specifications. Exp015 was a botched version of the successful Exp 016. This was the first successful reaction to produce DOPAL after several attempts that used sulfuric acid and a benzene extraction layer: Exp005, Exp008, Exp009, Exp010, Exp011, Exp012, Exp013. DOAPL was later made in a purer content in Exp025.
Ugi Reaction Procedure and monitoring the imine reversal
Solutions of an aldehyde and amine were made in an NMR solvent either MeOH-d4 or CDCl3 in the presence of a certain amount of 3A molecular sieves to remove water already in the solvent. The aldehyde and amine solutions were added and mixed together. The solutions usually turned a pale yellow. Reaction of the formation of the imine was monitored by HNMR over any given time period. Magnesium sulphate was added as well to dry the solution of the water, that forms during the imine formation, in an attempt to inhibit the reversal of the imine. After shaking the solution with the magnesium sulphate, the magnesium sulphate was allowed to settle and the solution was filtered. A boc protected acid solution and a tert-butylisocyanide solution were prepared also in the presence of sieves. Added the imine, acid and isocyanide solutions together and shook well. Solutions changed to a deep yellow to orange color. Reaction was again monitored by NMR. This reaction was done in two steps: first forming the imine and then adding the acid and isocyanide to form the Ugi product. The imine reaction was expected to go to completion as a one to one ratio and then to the Ugi product without any other interactions with the acid or isocyanide. Major experiments of this type included: Exp048, Exp051, Exp058, Exp066, Exp069, Exp076, Exp077, Exp080, Exp083, and Exp084.
Results
DOPAL
This was the first successful production of DOPAL. Here we can see the HMR(with integration) of 16A. The solubility of the product was good in methanol, but poor in methylene chloride. Here we have another HMR(w/o integration) with D2O added making 16B just to see the phenol protons exchange with the D2O (note: the scale is way off). See also Exp 016. A later experiment yielded a pure DOPAL product, see Exp025
Ugi Reaction Procedure and monitoring the imine reversal
The first part was the monitoring of the imine formation. The aldehyde, piperonal, and the amine, aniline, were mixed together and allowed to react over three days bringing the reaction to completion. The treatments with 3A molecular sieves and magnesium sulphate were done to remove water so when the acid and isocyanide were added, the imine reversal would not happen or at least be minimized. Here we have the HNMR of the imine after 3 days from Exp084-C. Calculations of the imine concentration can be found on the excel spreed sheet. See Exp084 for more spectra.
The next step was to add the acid and isocyanide. Here is an
Listed here is an excel sheet of the imine percent differences before and after the acid was added for all major reactions. Included also is the normalization of all major reactions with graphs. From this excel sheet the Figures I & II were obtained.
This is a graph of the % difference in imine concentration (taken before and directly after the addition of the acid, see the Discussion section for sample equation used) versus the mass of the 3A activated sieves used. The NMR solvents represented are both MeOH-d4 and CDCl3. Figure I
This is a graph of the % difference in imine concentration (taken before and directly after the addition of the acid) versus the mass of magnesium sulphate used for the imine portion of the experiment. Magnesium sulphate was not used for every experiment so only four experiments are represented here but every one of these experiments also employed activated sieves. Figure II
Discussion
DOPAL
The assignment of the major peaks for the H-NMR of 16A are: 9.57(CHO),8.96(phenol), 8.9(phenol), 6.5(aromatic), 6.6(aromatic), 6.7(aromatic), 3.5 (CH2). Peaks at 3.4 and 2.5 are DMSO-d6. Addition of D2O removes the 2 peaks near 9 ppm, supporting the phenolic assignments.7
Li et al in 1998 8 reported the H-NMR for DOPAL in DMSO-d6 as: 9.67(t, 1H, CHO), 7.27 (dd, 1H, aromatic), 7.2 (d, 1H, aromatic), 6.9 (d, 1H, aromatic), 3.5 (d, 2H, CH2). The peaks in the 6.9-7.3 ppm range are inconsistent with HMR spectra of 4-alkylcatechols, such as 4-methylcatechol, with the three aromatic hydrogens generally appearing in the 6.5-6.9 ppm range.
Since the HNMR assignments from this paper is inconsistent with the majority of other spectra of similar molecules, as well as 9Nobuhiro Fusetani et al Tetrahedron 1994, more than likely, the assignments made for our spectrum are correct and that the peaks reported by Li are erroneous. See Exp 016 for further spectra.
Ugi Reaction Procedure and monitoring the imine reversal
When determining the amount of a component, the integration of the component that is being compared to the imine shows up in the numerator of the equation:(X/(X+Y))*100(This is equation "A"). When the imine is compared to that component it takes the X variable in the equation. The numbers reported are percentages of the component converted which is the equation: 100-((X/(X+Y))*100) (This is equation "B"). For example: The aldehyde converted X% compared to the imine, this means that the aldehyde is in the X variable position and the imine is the Y variable position. This can be seen on the excel sheet as well which also has integration data which can also be obtained from the spectrum provided. The percent difference in the imine concentration, that was used for Figures I & II, is determined by the equation: ((ImA-ImB)/(ImA+ImB))*100 (This is equation "C"). Where ImA is the percentage determined by equation "A" before the acid was added and ImB is the percentage determined by equation "A" after the acid was added. The absolute value is not taken as a negative value would indicate further imine progression rather than reversal. Note For Exp051 there was an increase in the imine concentration directly after adding the acid making the value for equation "C" negative however, the acid reaction was monitored further and the imine gradually decreased. The end value of the imine concentration after the acid was added for this experiment is what was used for the value of ImB.
The imine reactions usually reach completion after 24 hours.
The percentages of imine, amine, and aldehyde were normalized to the component that went to completion, in some cases the amine,and in others the aldehyde and then plotted versus time. It seems that the decrease in the concentration of the imine is equivalent to the increase of concentration of the aldehyde for most of the experiments done. However the starting concentrations of most experiments are different and thus a direct comparison is difficult to produce. The normalized excel data is provided. The normalization calculation was done via Origin software.
A crystalline Ugi product10 did not form in any experiment done possibly because of the side reactions that were more favorable or because of a high Ksp for the corresponding Ugi product. However the peaks that appear around 5.8 and 5.88 and 6.1ppm possibly correspond to one NH group, chiral proton, and furan protons. These results of the enantiomeric proton are consistent with other results from Exp052 .
Conclusions
For DOPAL, the HNMR evidence of the crude ether wash shows that DOPAL can be made and isolated in 5-10% yield as a major product after 15 minutes of adrenaline decomposition in 85% phosphoric acid at 100-120 C. This material is not quite pure enough to be used without chromatography but as previously stated, a later experiment produced a pure DOPAL product Exp025.
It is clear that the imine was not prevented from fully reverting to the aldehyde and amine. There were however some experiments where the imine reverted only about 19% and less immediately after the addition of the acid. There does not seem to be any correlation to the percent reversal and the amount of sieves or amount of magnesium sulphate as the graphs show. No crystalline Ugi product was ever formed however some of the Ugi product was formed compared to previous NMR were the Ugi product was isolated.
Future Work
DOPAL can be made in a pure, low yielding quantity that, if the right conditions are found to keep it from oxidizing to the acid, can be sold commercially. It is unclear that the imine was completely stopped from reverting due to inconsistencies in prior experiments. The imine reversal is an interesting effect to study as well as the entire mechanism of this reaction. Reasons for why some Ugi products precipitate and others do not is also another area to explore. As of yet no Ugi products have been tested on inhibiting enoyl reductase so further exploration of possible inhibiting piperazines to malaria and other diseases can be done.
Hulme, C; Morrissette, Matthew M.; Volz Francis A.; and Burns Christopher J., The solution phase synthesis of diketopiperazine libraries via the Ugi reaction: Novel application of Armstrong's convertible isonitrile. Tetrahedron Letters 1998, 39(10), 1113-1116. http://dx.doi.org/10.1016/S0040-4039%2897%2910795-X
Robbins, Jay H.; Preparation and properties of p-hydroxyphenylacetaldehyde and 3-methoxy-4-hydroxyphenylacetaldehyde. Archives of Biochemistry and Biophysics, 114(3), 1966, 576-584. http://dx.doi.org/10.1016/0003-9861%2866%2990382-1
Li, Shu Wen; Spazianoa, Vincent T.; and Burke, William J.; Synthesis of a Biochemically Important Aldehyde, 3,4-Dihydroxyphenylacetaldehyde, Bio-organic Chemisty, 26(1), 1998, 45-50. http://dx.doi.org/10.1006/bioo.1998.1087
Synthesizing DOPAL, working the Ugi Reaction, and
Open Source Science
Abstract
Introduction
Experimental
DOPAL Procedure
Ugi Reaction Procedure and monitoring the imine reversal
Results
DOPAL
Ugi Reaction Procedure and monitoring the imine reversal
- The first part was the monitoring of the imine formation. The aldehyde, piperonal, and the amine, aniline, were mixed together and allowed to react over three days bringing the reaction to completion. The treatments with 3A molecular sieves and magnesium sulphate were done to remove water so when the acid and isocyanide were added, the imine reversal would not happen or at least be minimized. Here we have the HNMR of the imine after 3 days from Exp084-C. Calculations of the imine concentration can be found on the excel spreed sheet. See Exp084 for more spectra.
- The next step was to add the acid and isocyanide. Here is an
HNMR of the Ugi solution after 2 hrs 084D. Overlays of both parts of the reaction can be found on the experiment page.This is a graph of the % difference in imine concentration (taken before and directly after the addition of the acid, see the Discussion section for sample equation used) versus the mass of the 3A activated sieves used. The NMR solvents represented are both MeOH-d4 and CDCl3.
Figure I
This is a graph of the % difference in imine concentration (taken before and directly after the addition of the acid) versus the mass of magnesium sulphate used for the imine portion of the experiment. Magnesium sulphate was not used for every experiment so only four experiments are represented here but every one of these experiments also employed activated sieves.
Figure II
Discussion
DOPAL
Ugi Reaction Procedure and monitoring the imine reversal
Note For Exp051 there was an increase in the imine concentration directly after adding the acid making the value for equation "C" negative however, the acid reaction was monitored further and the imine gradually decreased. The end value of the imine concentration after the acid was added for this experiment is what was used for the value of ImB.
Conclusions
Future Work
Reference List
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